viruses
Review
Chloroviruses Have a Sweet Tooth
James L. Van Etten 1, *, Irina Agarkova 1 , David D. Dunigan 1 , Michela Tonetti 2 ,
Cristina De Castro 3 and Garry A. Duncan 4
1
2
3
4
*
Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska-Lincoln,
Lincoln, NE 68583-0900, USA; [email protected] (I.A.); [email protected] (D.D.D.)
Department of Experimental Medicine and Center of Excellence for Biomedical Research,
University of Genova Viale Benedetto XV/1, 16132 Genova, Italy; [email protected]
Department of Agricultural Sciences, University of Napoli, Via Università 100, 80055 Portici, NA, Italy;
[email protected]
Department of Biology, Nebraska Wesleyan University, Lincoln, NE 68504-2796, USA;
[email protected]
Correspondence: [email protected]; Tel.: +1-402-472-3168
Academic Editors: Tessa E.F. Quax, Matthias G. Fischer and Laurent Debarbieux
Received: 17 March 2017; Accepted: 14 April 2017; Published: 22 April 2017
Abstract: Chloroviruses are large double-stranded DNA (dsDNA) viruses that infect certain isolates
of chlorella-like green algae. They contain up to approximately 400 protein-encoding genes and
16 transfer RNA (tRNA) genes. This review summarizes the unexpected finding that many of the
chlorovirus genes encode proteins involved in manipulating carbohydrates. These include enzymes
involved in making extracellular polysaccharides, such as hyaluronan and chitin, enzymes that
make nucleotide sugars, such as GDP-L-fucose and GDP-D-rhamnose and enzymes involved in the
synthesis of glycans attached to the virus major capsid proteins. This latter process differs from that
of all other glycoprotein containing viruses that traditionally use the host endoplasmic reticulum and
Golgi machinery to synthesize and transfer the glycans.
Keywords: chloroviruses; giant viruses; hyaluronan synthesis; chitin synthesis; nucleotide sugar
synthesis; glycan synthesis; glycan structures; glycoproteins
1. Introduction
In discussing enzymes involved in manipulating carbohydrates, one usually does not consider
viruses to play a role in this important subject. However, as described in this review, chloroviruses
(family Phycodnaviridae) that infect certain isolates of single-celled, eukaryotic chlorella-like green
algae are an exception to this process because they encode enzymes involved in making
extracellular polysaccharides, nucleotide sugars and the synthesis of glycans attached to their major
capsid glycoproteins.
The plaque-forming chloroviruses are large icosahedral (190 nm in diameter), double-stranded
DNA (dsDNA)-containing viruses (genomes of 290 to 370 kb) with an internal lipid membrane.
They exist in inland waters throughout the world with titers occasionally reaching thousands of
plaque-forming units (PFU) per mL of indigenous water. Known chlorovirus hosts, which are
normally endosymbionts and are often referred to as zoochlorellae [1,2], are associated either with the
protozoan Paramecium bursaria, the coelenterate Hydra viridis or the heliozoan Acanthocystis turfacea [3–6].
Four such zoochlorellae and their viruses are Chlorella variabilis NC64A and its viruses (referred
to as NC64A viruses), Chlorella variabilis Syngen 2–3 and its viruses (referred to as Osy viruses),
Chlorella heliozoae SAG 3.83 and its viruses (referred to as SAG viruses) and Micractinium conductrix Pbi
and its viruses (referred to as Pbi viruses). The zoochlorellae are resistant to virus infection when they
are in their symbiotic relationship, because the viruses have no way of reaching their hosts.
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The genomes of 43 chloroviruses infecting these four hosts have been sequenced, assembled and
annotated [6–11]. Collectively, the viruses encode genes from 643 predicted protein families; however,
any given chlorovirus only has 330 to 416 protein-encoding genes (PEGs). Thus, the genetic diversity
among these viruses is large, and many of the proteins are unexpected for a virus. With the exception
of homologs solely in other chlorovirus members, about 50% of their PEGs do not match anything in
the databases.
The prototype chlorovirus Paramecium bursaria chlorella virus type 1 (PBCV-1) is an NC64A
virus [12]. PBCV-1 is an icosahedron (190 nm in diameter) with a spike-like structure at one vertex
and a few external fibers that extend from some of the viral capsomeres [5,13]. The outer capsid
layer covers a single lipid bilayered membrane, which is essential for infection. The PBCV-1 major
capsid protein (named Vp54) is a glycoprotein, and three Vp54s form a trimeric capsomere, which has
pseudo-six-fold symmetry. A proteomic analysis of PBCV-1 virions revealed that the virus contains
148 virus-encoded proteins and at least one host-encoded protein [10]. The PBCV-1 genome is a linear
~331-kb, non-permuted dsDNA molecule with covalently-closed hairpin termini. Identical ~2.2-kb
inverted repeats flank each 35-nucleotide-long, incompletely base-paired, covalently closed hairpin
loop [14,15]. The remainder of the PBCV-1 genome contains primarily single-copy DNA that encodes
~416 putative proteins and 11 transfer RNAs (tRNAs) [5]. The G + C content of the PBCV-1 genome
is 40%; in contrast, its host nuclear genome is 67% G + C. PBCV-1 and other chlorovirus genomes
contain methylated bases, which occur in specific DNA sequences. The methylated bases are part of
chlorovirus-encoded DNA restriction and modification systems [16].
As the title of this review indicates, many of the chlorovirus genes encode enzymes involved
in various aspects of carbohydrate metabolism. We have listed putative chlorovirus genes involved
in carbohydrate metabolism, which are encoded by the 43 chloroviruses whose genomes have been
sequenced, in Table 1, Table 2 and Table 4. Recombinant proteins have been produced from some of
these genes, and the proteins have been characterized (indicated in bold in the tables). When some of
the genes were initially cloned and the recombinant proteins characterized, the genes were hybridized
to many other chlorovirus genomes by dot blots to determine the distribution of the genes. Because of
the large number of viruses, these experimental results are not included in the tables, unless the virus
genome was subsequently sequenced.
Table 1. Chlorovirus encoded enzymes involved in the synthesis of polysaccharides.
Host
Viruses
NC64A
AN69C
AR158
CviK1
CvsA1
IL-3A
IL-5-2s1
KS-1B
MA-1D
MA-1E
NE-JV-4
NY-2B
NY2A
NYs-1
PBCV-1
SYN
OSY-NE-5
HAS 1
102R
CHS 2
CBP 3
390R
C418R
365R
375R
395L, 438L
C423L, C475L
370L, 414L
380L, 427L
386L, 432L
503L, 562L
314L, 360L
367L, 472L
407L, 451L
390L
542L, 484L
B480L
360R, 495L, 555L
A333L, A348Dl, A383R
134L
485L
362R
113L
B139R, B472R
a
137R
A098R
038R
167L, 184L
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Table 1. Cont.
Host
Viruses
Pbi
AP110A
CVA-1
CVB-1
CVG-1
CVM-1
CVR-1
CZ-2
Can18-4
FR483
FR5L
MT325
NE-JV-1
NW665
OR0704.2.2
SAG
ATCV-1
Br0604L
Can0610SP
Canal-1
GM0701
MN0810
MO0605SPH
NE-JV-2
NE-jv-3
NTS-1
OR0704.3
TN603
W10606
HAS 1
CHS 2
CBP 3
152L, 175R
150L, 169R
177L
828R
834R
791R
792R
832R
838R
798R
839R
N690R
797R
M701R
734L
821R
804R
146R
165L, 186R
163R
N124R
151L
M128R
278R, 282R
133R
116R
834R
746R
852R
087R, 900R
893R
869R
Z734R
431R
438R, 442R
405R
436R
466R, 468R, 531L
435R
462R
431R
461R, 463R, 529L
431R
425R
457R
1 Hyaluronan synthase; 2 chitin synthase; 3 chitin binding proteins, except for the chitinase proteins reported in
Table 5; a the recombinant protein has the predicted activity. The numbers refer to the protein names, and the R and
L refer to the strand orientation.
2. Chlorovirus Encoded Polysaccharide Synthesizing Enzymes
Three PBCV-1 encoded enzymes are involved in the synthesis of the extracellular matrix
polysaccharide hyaluronan (also referred to as hyaluronic acid), including glycosyltransferase Class
I hyaluronan synthase (HAS; Table 1) [17,18]. Until the has gene (a098r) was discovered in PBCV-1,
hyaluronan was only thought to occur in vertebrates and a few pathogenic bacteria, where it forms
an extracellular capsule, presumably to avoid the immune system [19,20]. Hyaluronan is an essential
constituent of the extracellular matrix in vertebrates and consists of ~10,000 or more alternating
β-1,4-glucuronic acid (GlcA) and β-1,3-N-acetylglucosamine (GlcNAc) residues. Typically, the HAS
enzyme is located on the inner surface of the plasma membrane. The newly-synthesized hyaluronan
then moves through the membrane and cell wall to the extracellular matrix.
PBCV-1 also encodes two enzymes involved in the biosynthesis of hyaluronan precursors,
glutamine:fructose-6-phosphate amidotransferase (GFAT, gene a100r) and UDP-glucose dehydrogenase
(UDP-GlcDH, gene a609l; Table 2) [21]. All three PBCV-1 genes involved in hyaluronan synthesis
are expressed early during virus infection, and all three transcripts decrease significantly by 60 min
post-infection (PI) [18,21]. However, these three genes do not function like an operon, although
two of the genes, a98r and a100r, are adjacent to one another and are co-linear in the PBCV-1
genome. In contrast, a609l is located ~240 kb away and is transcribed in the opposite orientation [17].
The identification of these three genes led to the discovery that hyaluronan lyase-sensitive hair-like
fibers begin to accumulate on the surface of PBCV-1-infected host cells by 15 min PI. By 4 h PI, the
infected cells are covered with a dense fibrous hyaluronan network (Figure 1) [18].
Viruses 2017, 9, 88
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Table 2. Chlorovirus encoded enzymes involved in sugar metabolism.
Host
Viruses
GFAT 1
UDP-GlcDH 2
GMD 3
GMER 4
109R
C132R
105R
064R
104R
130R
384R, 487R
C413R, C729L
359R, 662L
368R
375R, 685L
492R, 858L
NC64A
AN69C
AR158
CviK1
CvsA1
IL-3A
IL-5-2s1
KS-1B
MA-1D
MA-1E
NE-JV-4
NY-2B
NY2A
NYs-1
PBCV-1
481L
116R
355R, 838L
396R
B143R
143R
a A100R
473R, 836L
B465R
483R, 846L
a A609L
129R
C155R
122R
083R
126R
106L
066R
456L
134R
131R
185R
B163R
167R
a A118R
OSY-NE-5
039R
045R
AP110A
CVA-1
CVB-1
CVG-1
CVM-1
CVR-1
CZ-2
Can18-4
FR483
FR5L
071R
056R
071R
050R
069R
062R
059R
061R
N035R
087R
M036R,
M037R
081R
046R
062R
SYN
Pbi
MT325
NE-JV-1
NW665
OR0704.2.2
UGD 5
AT 6
D-LD 7
ADP-RGH 8
334L
C344L
312L
321L
326L
417L
261L
284L
355L
340L
408L
B395L
404L
a A295L
739L
C767L
742L
716L
726L
896L
643L
872L
806L
740L
881L
B853L
879L
A654L
055R
A053R
139L
340L
015R
308R
146L
144L
172L
812L
159L
151L
718L
859L
N712L
145L
893R
900R
856R
857R
893R
906R
865R
908R
N747R
863R
053L
040L
056L
205R
221R
M719L
M758R
M026L
291R
846L
722L
861R
889R
862R
810L
FRD 9
055R
131R
051R
045R
064R
052L
046L
048L
048L
222R
210R
917L
212R
N170R
076L
189R
051L
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Table 2. Cont.
Host
SAG
1
Viruses
ATCV-1
Br0604L
Can0610SP
Canal-1
GM0701
MN0810
MO0605SPH
NE-JV-2
NE-JV-3
NTS-1
OR0704.3
TN603
W10606
GFAT 1
887L
UDP-GlcDH 2
Z571L
667L, 839R
687L
605L, 751R
664L, 856R
720L, 904R
656L
708L
679L
714L, 898R
676L
659L, 873R
679L
2
GMD 3
a
Z804L
934L
965L
847L
954L
992L
897L
981L
935L
1012L
960L
966L
916L
GMER 4
UGD 5
AT 6
D-LD 7
Z282L
332L
338L
329L
337L
365L
341L
367L
332L
378L
335L
326L
360L
Z544R
631R
658R
576R
629R
689R
625R
672R
648R
681R
639R
625R
651R
Z147L
173L
170L
188L
180L
204L
181L
186L
175L
188L
179L
179L
185L
Z295L
350L
355L
343L
354L
379L
359L
383L
347L
391L
354L
342L
375L
3
4
ADP-RGH 8
FRD 9
898L
886R
943R
981R
962R
Glutamine-fructose-6-phosphate aminotransferase; UDP-glucose-6-dehydrogenase; GDP-D-mannose dehydratase; GDP-4-keto-6-deoxy-D-mannose epimerase/reductase
(=GDP-L-fucose synthase 2); 5 UDP-D-glucose 4,6-dehydratase, 6 acetyltransferase; 7 D-lactate dehydrogenase; 8 ADP-ribosylglycohydrolase; 9 fumarate reductase; a the recombinant
proteins have the predicted activities. The numbers refer to the protein names, and the R and L refer to the strand orientation.
Viruses 2017, 9, 88
Viruses 2017, 9, 88 6 of 23
6 of 23 C
C
Cyto C
Cyto A
B
Cyto C Figure
Location of of hyaluronan hyaluronan on on the the surface surface of of infected
Figure 1.
1. Location infected Chlorella
Chlorella variabilis
variabilis NC64A
NC64A cells
cells and
and ultrastructural changes in the algal cell wall after chlorovirus Paramecium bursaria chlorella virus type
ultrastructural changes in the algal cell wall after chlorovirus Paramecium bursaria chlorella virus type 1 1 (PBCV-1) infection. The figure shows the cross-sections of (A) the surface of the uninfected cells;
(PBCV‐1) infection. The figure shows the cross‐sections of (A) the surface of the uninfected (B) cells at 4 h post-infection (PI); and (C) cells at 4 h PI that were treated with hyaluronan lyase. Note
cells; (B) cells at 4 h post‐infection (PI); and (C) cells at 4 h PI that were treated with hyaluronan that after treatment with hyaluronan lyase, the cell surface resembles the surface of uninfected cells. C is
lyase. Note that after treatment with hyaluronan lyase, the cell surface resembles the surface of the cell wall, and Cyto is cytoplasm. Micrographs were taken from Graves et al. [18] with permission.
uninfected cells. C is the cell wall, and Cyto is cytoplasm. Micrographs were taken from Graves et al. [18] with permission. Three additional enzymes are needed to convert glucosamine-6-phosphate (GlcN-6P) to
UDP-N-acetylglucosamine
(UDP-GlcNAc), and these enzymes (EC2.3.1.4, EC5.4.2.3, EC2.7.7.23) are
The has gene that encodes hyaluronan synthase is present in 12 of the 43 chloroviruses isolated encoded
by the host [22]. This is not surprising because the host NC64A cell wall is predicted to
from diverse geographical regions, including 5 NC64A viruses, 6 Pbi viruses and 1 Osy virus (Table 1). In contain
chitin, which is a polymer of GlcNAc residues, and so, the alga must encode these enzymes.
contrast, the udp‐glcdh gene is present in 40 of the 43 viruses, 14 of which have two copies of the gene, The has gene that encodes hyaluronan synthase is present in 12 of the 43 chloroviruses isolated
while the gfat gene is present in 27 chloroviruses, including 11 of the 14 NC64A viruses, all 14 Pbi from
diverse geographical regions, including 5 NC64A viruses, 6 Pbi viruses and 1 Osy virus (Table 1).
viruses, one of the 13 SAG viruses and the only Osy virus that has been sequenced (Table 2). Both of In
contrast, the udp-glcdh gene is present in 40 of the 43 viruses, 14 of which have two copies of the
these latter two genes are present in all of the 12 viruses that have a has gene, except for the one Osy gene,
while the gfat gene is present in 27 chloroviruses, including 11 of the 14 NC64A viruses, all 14 Pbi
virus that lacks a udp‐glcdh gene. viruses,
one of the 13 SAG viruses and the only Osy virus that has been sequenced (Table 2). Both of
Surprisingly, 19 of the 31 chloroviruses that lack a has gene have a gene encoding a chitin synthase these
latter
two an genes
are present
in homopolymer all of the 12 viruses
that have a has gene,residues, except for
one Osy
(CHS). Chitin, insoluble linear of β‐1,4‐linked‐GlcNAc is the
a common virus
that lacks a udp-glcdh gene.
component of insect exoskeletons, shells of crustaceans and fungal cell walls [23]. Chitin is rare in Surprisingly, 19 of the 31 chloroviruses that lack a has gene have a gene encoding a chitin synthase
algal cell walls, although it has been reported to exist in some green algae [24]. Like the has gene, the (CHS).
Chitin, an insoluble linear homopolymer of β-1,4-linked-GlcNAc residues, is a common
chs gene is expressed as early as 10 min PI and peaks at 20–40 min PI, and the transcript disappears component
of insect exoskeletons, shells of crustaceans and fungal cell walls [23]. Chitin is rare in
at 120–180 min PI. Furthermore, cells infected with chs‐containing viruses produced chitin fibers on algal
cell walls, although it has been reported to exist in some green algae [24]. Like the has gene, the
the external surface of their hosts [25]. As discussed below, many of the chloroviruses also encode chs
gene is expressed as early as 10 min PI and peaks at 20–40 min PI, and the transcript disappears
chitinases and chitosanases. at 120–180
min PI. Furthermore, cells infected with chs-containing viruses produced chitin fibers on
At least one chlorovirus, CVK2, has replaced the PBCV‐1 has gene with a 5‐kb region containing the
external surface of their hosts [25]. As discussed below, many of the chloroviruses also encode
chs, udp‐gdh2 (a gene encoding a second UDP‐GlcDH) and two other ORFs [26]. Therefore, at least chitinases
and chitosanases.
some chloroviruses have changed from HAS viruses to CHS viruses or vice versa, by swapping genes. At
least
one chlorovirus,
CVK2,
has
replaced
thechs PBCV-1
gene
with one a 5-kb
containing
Two NC64A chloroviruses have both has and genes, has
and at least of region
them forms both chs,
udp-gdh2
(a
gene
encoding
a
second
UDP-GlcDH)
and
two
other
ORFs
[26].
Therefore,
at
least
hyaluronan and chitin on the surface of their infected cells [25,27]. Finally, 12 chloroviruses lack both some
chloroviruses have changed from HAS viruses to CHS viruses or vice versa, by swapping genes.
genes, and no extracellular polysaccharides are formed on the surface of cells infected with at least Two NC64A chloroviruses have both has and chs genes, and at least one of them forms both
one of these viruses [18]. The fact that many chloroviruses encode enzymes involved in extracellular hyaluronan
and chitin on the surface of their infected cells [25,27]. Finally, 12 chloroviruses lack both
polysaccharide biosynthesis suggests that the polysaccharides, which require a large expenditure of genes,
and no extracellular polysaccharides are formed on the surface of cells infected with at least
ATP for their synthesis, are important in the virus life cycles. However, the extracellular hyaluronan one
of these viruses [18]. The fact that many chloroviruses encode enzymes involved in extracellular
does not play an obvious role in the interaction between PBCV‐1 and its algal host because neither polysaccharide
biosynthesis suggests that the polysaccharides, which require a large expenditure of
plaque size nor plaque numbers were altered by including either hyaluronidase or free hyaluronan ATP
for their synthesis, are important in the virus life cycles. However, the extracellular hyaluronan
in the top agar of the PBCV‐1 plaque assay [17]. does The not play
obvious
role inin thesynthesizing interaction between
PBCV-1
and
its algalbeen host obtained because neither
three angenes involved hyaluronan have probably rather plaque
size nor plaque numbers were altered by including either hyaluronidase or free hyaluronan in
recently in evolutionary terms because the coding portions of the PBCV‐1 gfat and udp‐glcnc genes the
top agar of the PBCV-1 plaque assay [17].
are 44% G + C, while the has gene is 46.7% G + C. In contrast, PBCV‐1, as well as all the NC64A viruses, The three genes involved in synthesizing hyaluronan have probably been obtained rather recently
have a 40% G + C content [11,21]. in evolutionary
the coding
portions
of the the PBCV-1
gfat and udp-glcnc
genesthese are Currently, terms
it is because
not known how or why chloroviruses acquired polysaccharide‐synthesizing genes. We have considered the following possible evolutionary advantages for acquiring these genes: (1) the polysaccharides prevent infection by a second chlorovirus; (2) they cause the infected cells to clump with uninfected host cells, thus increasing the probability that Viruses 2017, 9, 88
7 of 23
44% G + C, while the has gene is 46.7% G + C. In contrast, PBCV-1, as well as all the NC64A viruses,
have a 40% G + C content [11,21].
Currently, it is not known how or why the chloroviruses acquired these polysaccharide-synthesizing
genes. We have considered the following possible evolutionary advantages for acquiring these genes:
(1) the polysaccharides prevent infection by a second chlorovirus; (2) they cause the infected cells to clump
with uninfected host cells, thus increasing the probability that progeny viruses can infect healthy host
cells; (3) they prevent paramecia from taking up infected algal cells, (4) the chloroviruses have another
host in nature, and this other host is attracted to or binds to hyaluronan or chitin on virus-infected
algae, which would facilitate progeny-virus infections; or (5) polysaccharides increase the functional
diameter of the infected cell, which might facilitate consumption by a predator. This could aid virus
movement in the water column. In regards to the first possibility, it is known that attachment of
other viruses to PBCV-1-infected cells at 4 h PI is inhibited when the external surface of the host is
covered with hyaluronan fibers [18]. However, this is unlikely to be the explanation for the presence
of hyaluronan because normally the host, C. variabilis NC64A, is only infected by one virus, and this
restriction occurs in the first few min PI [28,29]. In regards to the second possibility, host cells often
clump shortly after infection, and this phenomenon, which does not always occur, could be due to
hyaluronan production. The last three possibilities have not been explored experimentally.
We have experimentally tried to address the question: does the presence of hyaluronan and/or
chitin on the exterior surface of the host cell wall confer an evolutionary advantage to a virus that has
one or both of these genes? To answer the question, chlorella cells were co-infected with combinations
of chloroviruses that: (1) have both genes; (2) only have the has gene; (3) only have the chs gene; and
(4) lack both genes. The resulting lysates were then added to fresh cells and allowed to replicate and
lyse. After five passages, progeny viruses were plaqued, and 20 plaques were randomly picked to
determine if one virus type dominated. However, after repeating these experiments several times, no
consistent pattern was obtained [30].
To ideally conduct this experiment, one would like to either add the chs gene to the PBCV-1
genome so that both genes are present, replace the has gene with the chs gene or remove the has
gene so that PBCV-1 lacked both genes. Unfortunately, this experimental protocol is currently not
possible because procedures are not available for reverse genetic manipulation of chlorovirus genomes.
Therefore, in the experiments described above, viruses were selected that had the desired properties
and also had similar growth kinetics as PBCV-1.
In addition to not knowing why the chloroviruses acquired the has and chs genes, another question
is: how are the newly-forming hyaluronan and/or chitin fibers moved through the membrane and
the complex cell wall to the exterior of the algal host from the plasma membrane? This phenomenon
would appear to be equivalent to pushing a thread through a furnace filter. One would expect the
polysaccharide fibers to bunch up underneath the cell wall. In fact, this happened when the viral
has gene was expressed in cultured tobacco cells [31]. Could a pilot protein(s) that is attached to the
leading end of the polymer guide the hyaluronan chain through the wall?
3. Chlorovirus Encoded Nucleotide Sugar Metabolism Enzymes
Many chloroviruses also encode enzymes involved in nucleotide sugar metabolism, as well as
other sugar metabolic enzymes (Table 2). Two enzymes encoded by all of the NC64A, SAG and
Syn chloroviruses, GDP-D-mannose 4,6 dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose
epimerase reductase (GMER) (Table 2), comprise a highly-conserved pathway in bacteria, plants
and animals that converts GDP-D-mannose to GDP-L-fucose (Figure 2) [32]. Fucose is found in
glycoconjugates of many organisms, where it often plays a fundamental role in cell-cell adhesion and
recognition [33]. The Pbi chloroviruses lack both gmd and gmer genes (Table 2) even though the glycans
attached to the major capsid protein from the three evaluated Pbi viruses have fucose [34,35].
In vitro reconstruction of the pathway using recombinant PBCV-1 GMD and GMER proteins
resulted in the synthesis of GDP-L-fucose as expected. Unexpectedly, however, the PBCV-1 GMD
Viruses 2017, 9, 88
8 of 23
also catalyzed the NADPH-dependent reduction of the intermediate GDP-4-keto-6-deoxy-D-mannose,
to form GDP-D-rhamnose. That is, the enzyme has two activities, and both sugars are produced in
the infected cell [32]. The PBCV-1 recombinant GMD has another property that is unusual. Unlike
recombinant GMDs from many other organisms, the viral encoded enzyme is very stable when stored
at either 4 ◦ C or −20 ◦ C [32]. The PBCV-1 GMD enzyme was crystalized, and the structure resembles
Viruses 2017, 9, 88 8 of 23 other GMDs [36].
Figure 2. Scheme of the biosynthesis of GDP‐
Figure 2. Scheme of the biosynthesis of GDP-LL‐fucose and GDP‐D‐rhamnose. PBCV‐1 GDP‐
-fucose and GDP-D-rhamnose. PBCV-1 GDP-D
D‐mannose -mannose
4,6 intermediate 4,6 dehydratase dehydratase (GMD) (GMD) catalyzes catalyzes both both the the dehydration dehydration of of GDP‐
GDP-D
D‐mannose -mannose to to the the intermediate
GDP‐4‐keto‐6‐deoxy‐
the NADPH-dependent
NADPH‐dependent reduction latter compound
compound to
to GDP-4-keto-6-deoxy-DD‐mannose -mannose and and the
reduction of of this this latter
++
D
‐rhamnose. NADP
serves as the cofactor for GMD during the internal oxidoreduction reaction GDP‐
GDP-D-rhamnose. NADP serves as the cofactor for GMD during the internal oxidoreduction reaction
involved the dehydration
dehydration process.
process. The epimerization and the NADPH-dependent
NADPH‐dependent reduction the involved in in the
The epimerization
and the
reduction of of the
by PBCV-1
PBCV‐1 GDP-4-keto-6-deoxy-D-mannose
GDP‐4‐keto‐6‐deoxy‐D‐mannose 4‐keto 4-keto group group leading leading to to GDP‐
GDP-LL‐fucose -fucoseare are carried carried out out by
epimerase reductase (GMER). Figure was taken from Tonetti et al. [32] with permission. epimerase reductase (GMER). Figure was taken from Tonetti et al. [32] with permission.
A recombinant GMD protein encoded by another chlorovirus, Acanthocystis turfacea chlorella A recombinant GMD protein encoded by another chlorovirus, Acanthocystis turfacea chlorella
virus 1 (ATCV‐1), which has 53% amino acid identity with the PBCV‐1 GMD, was also characterized virus 1 (ATCV-1), which has 53% amino acid identity with the PBCV-1 GMD, was also characterized
because the amino acid differences between the two enzymes suggested they might have slightly different because the amino acid differences between the two enzymes suggested they might have slightly
properties. In fact, the ATCV‐1 GMD does not form GDP‐D‐rhamnose, and so, it lacks the second different properties. In fact, the ATCV-1 GMD does not form GDP-D-rhamnose, and so, it lacks the
enzyme activity [37]. Both GMD enzymes bound NADPH tightly, and this association was essential second enzyme activity [37]. Both GMD enzymes bound NADPH tightly, and this association was
for the stabilization and function of both enzymes, even though NADP+ is the co‐enzyme required to essential for the stabilization and function of both enzymes, even though NADP+ is the co-enzyme
initiate the GMD catalytic cycle. Phylogenetic analyses established that the PBCV‐1 GMD is the most required to initiate the GMD catalytic cycle. Phylogenetic analyses established that the PBCV-1 GMD
evolutionarily diverged of all the GMDs, whereas the ATCV‐1 GMD was in a clade of bacterial is the most evolutionarily diverged of all the GMDs, whereas the ATCV-1 GMD was in a clade of
GMDs [37]. bacterial GMDs [37].
The GMER enzymes from PBCV‐1 and ATCV‐1 have 63% amino acid identity to each other and The GMER enzymes from PBCV-1 and ATCV-1 have 63% amino acid identity to each other
phylogenetically are more similar to one another and to other GMERs than are the two GMDs. and phylogenetically are more similar to one another and to other GMERs than are the two GMDs.
The possible evolutionary consequences of these differences have been discussed previously [37]. The possible evolutionary consequences of these differences have been discussed previously [37].
Both fucose and rhamnose are constituents of the glycans attached to the PBCV‐1 and ATCV‐1 major Both fucose and rhamnose are constituents of the glycans attached to the PBCV-1 and ATCV-1 major
capsid proteins (see below). However, the PBCV‐1 glycan contains three rhamnose residues, with capsid proteins (see below). However, the PBCV-1 glycan contains three rhamnose residues, with
one in the D‐configuration, whereas only one with L‐configuration is present in the ATCV‐1 glycan. one in the D-configuration, whereas only one with L-configuration is present in the ATCV-1 glycan.
Perhaps there was enough natural selection pressure on the PBCV‐1 GMD gene to evolve to Perhaps there was enough natural selection pressure on the PBCV-1 GMD gene to evolve to synthesize
synthesize GDP‐D‐rhamnose, whereas the ATCV‐1 GMD did not face this pressure. GDP-D-rhamnose, whereas the ATCV-1 GMD did not face this pressure.
ATCV‐1 and all of the SAG viruses, however, encode another enzyme, UDP‐D‐glucose ATCV-1 and all of the SAG viruses, however, encode another enzyme, UDP-D-glucose
4,6‐dehydratase (UGD), that is one of two enzymes involved in the synthesis of L‐rhamnose [38], and 4,6-dehydratase (UGD), that is one of two enzymes involved in the synthesis of L-rhamnose [38], and
this enzyme may contribute to rhamnose synthesis. The PBCV‐1 host chlorella, which is closely this enzyme may contribute to rhamnose synthesis. The PBCV-1 host chlorella, which is closely related
related to the ATCV‐1 host chlorella, encodes the second enzyme in the rhamnose pathway [22], and to the ATCV-1 host chlorella, encodes the second enzyme in the rhamnose pathway [22], and so the
so the host is predicted to be able to synthesize the rhamnose required for ATCV‐1 glycan synthesis. host is predicted to be able to synthesize the rhamnose required for ATCV-1 glycan synthesis.
4. Unusual Attachment of Glycans to the Chlorovirus Major Capsid Proteins Structural proteins of many viruses, such as rhabdoviruses, herpesviruses, poxviruses and paramyxoviruses, are glycosylated. Glycans contribute to the protease resistance and the antigenicity of these viruses. Most virus glycans are linked to Asn in the protein via N‐acetylglucosamine, although Viruses 2017, 9, 88
9 of 23
4. Unusual Attachment of Glycans to the Chlorovirus Major Capsid Proteins
Structural proteins of many viruses, such as rhabdoviruses, herpesviruses, poxviruses and
paramyxoviruses, are glycosylated. Glycans contribute to the protease resistance and the antigenicity
of these viruses. Most virus glycans are linked to Asn in the protein via N-acetylglucosamine,
although some viruses also have O-linked glycans attached to either Ser or Thr residues via an amino
sugar, usually N-acetylglucosamine or N-acetylgalactosamine. Typically, viruses use host-encoded
glycosyltransferases and glycosidases located in the endoplasmic reticulum (ER) and Golgi apparatus
to add and remove N-linked sugar residues from virus glycoproteins either co-translationally or
shortly after translation of the protein. This post-translational processing aids in protein folding,
progression in the secretory pathway and in the regulation of host-virus interactions [39–42]. After
folding the protein, virus glycoproteins are transported by host-sorting and membrane-transport
functions to virus-specified regions in host membranes where they displace host glycoproteins.
Progeny viruses then bud through these virus-specific target membranes, which is usually the final
step in the assembly of infectious viruses. Thus, nascent viruses only become infectious after budding
through the membrane, usually the plasma membrane, as they exit the cell. Consequently, the glycan
portion of virus glycoproteins is host-specific. The theme that emerges from these viruses is that virus
glycoproteins are synthesized and glycosylated by the same processes as host glycoproteins. Therefore,
the only way to alter virus protein glycosylation is to either grow the virus in a different host or have a
mutation that alters the virus protein glycosylation site.
Unlike the process described above, glycosylation of the chlorovirus major capsid proteins
differs from that scenario because the viruses encode most, if not all, of the machinery for the
process. In addition, the process occurs in the cytoplasm. The conclusion that the chlorovirus PBCV-1
major capsid protein (Vp54, gene a430l) is glycosylated by a different mechanism than that used by
other characterized viruses originally arose from antibody studies [43]. Rabbit polyclonal antiserum
prepared against intact PBCV-1 particles inhibited virus plaque formation by agglutinating the virions.
However, spontaneously-derived, antiserum-resistant, plaque-forming variants of PBCV-1 occurred
at a frequency of 10−5 –10−6 . At the time of the 1993 publication, these antiserum-resistant variants
fell into four serologically-distinct classes; two additional antigenic variants have subsequently been
isolated for a total of six variants (Table 3). Polyclonal antisera prepared against members of each of
these antigenic classes react predominately with the Vp54 equivalents from the viruses in the class
used for the immunization. Each of the Vp54 proteins from the antigenic variants migrated faster on
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) than those of the strains from
which they were derived, indicating a lower molecular weight. However, all of the de-glycosylated
Vp54 proteins migrated at the same rate on SDS-PAGE, indicating that the differences resided in the
size of the attached glycans. In addition, the nucleotide sequence of the a430l gene in each of the
variants was identical to the wild-type gene, which verified that the polypeptide portion of Vp54
was not altered in the mutants. Western blot analyses of Vp54 proteins isolated from the variants,
before and after removing the glycans with trifluoromethane-sulfonic acid or altering the glycan with
periodic acid, also supported the notion that the antigenic variants reflected differences in the Vp54
glycans, not the Vp54 polypeptide [43].
All of the glycan antigenic variants form plaques on their C. variabilis NC64A host, so one can
infer that the glycans are not directly involved in virus infection and virus replication. However,
anecdotal evidence suggests that the glycans are important in virus stability because the variants with
the smallest glycans do not remain infectious in storage as long as wild-type virus.
Additional observations supported the concept that PBCV-1 Vp54 glycosylation was unusual:
(1) unlike viruses that acquire their glycoproteins(s) by budding through a plasma membrane, which
results in infectious particles, plaque-forming PBCV-1 particles accumulate inside the host 30–40 min
before virus release [47]; (2) all of the antigenic variants were grown in the same host so the glycan
differences are not due to the host; (3) polyclonal antibodies to Vp54, the major capsid protein, do not
react with host glycoproteins; (4) the Vp54 protein lacks an ER and Golgi signal peptide; (5) unlike
Viruses 2017, 9, 88
10 of 23
most glycoproteins that exhibit size micro-heterogeneity, PBCV-1 Vp54 appears homogeneous on
SDS-PAGE; in addition, mass spectrometry analysis only revealed one satellite peak that differed from
the main peak by 140 Da, the approximate weight of either one arabinose or xylose residue [46]; and
(6) the ability to easily crystallize Vp54 as a homotrimer provided additional evidence that the protein
is essentially homogeneous [48,49].
Table 3. PBCV-1 antigenic variants that affect the molecular weight of the major capsid glycoprotein.
Antisera Classes
Class a
Label b
Predicted MW (kDa) c
SDS-PAGE Estimates (kDa) d
+
Wild-type
54.1
54
10 of 23 CME6
54
not determined
A
P91
52.8
53
Additional observations supported the concept that PBCV‐1 Vp54 glycosylation was unusual: E
EPA-15
52.8
not determined
(1) unlike viruses that acquire their glycoproteins(s) by budding through a plasma membrane, which B
EPA-2
51.6
52
results in infectious particles, plaque‐forming PBCV‐1 particles accumulate inside the host 30–40 min C
E1L3
51.1
51
before virus release [47]; (2) all of the antigenic variants were grown in the same host so the glycan D
P1L6
50.5
50.5
Viruses 2017, 9, 88 F
differences are not due to the host; (3) polyclonal antibodies to Vp54, the major capsid protein, do not Listed
in order of predicted molecular weight based on nuclear magnetic resonance (NMR) analysis
react with host glycoproteins; (4) the Vp54 protein lacks an ER and Golgi signal peptide; (5) unlike (De Castro
al., [44,45]); b representative
mutant
strain label; c thePBCV‐1 gene encoding
the PBCV-1
major capsid
protein
most etglycoproteins that exhibit size micro‐heterogeneity, Vp54 appears homogeneous on d Graves et al. [46]. MW, molecular weight.
(a430l) SDS‐PAGE; is wild-type in
sequence
and
does
not
vary
among
antisera
classes;
in addition, mass spectrometry analysis only revealed one satellite peak that differed a
from the main peak by 140 Da, the approximate weight of either one arabinose or xylose residue [46]; and (6) the ability to easily crystallize Vp54 as a homotrimer provided additional evidence that the Evidence that the N-linked Vp54 glycans are not attached to the Vp54 protein by a traditional
protein is essentially homogeneous [48,49]. N-linkage was initially obtained from the X-ray crystal structure of the protein. The structure revealed
Evidence that the N‐linked Vp54 glycans are not attached to the Vp54 protein by a traditional that the protein
had four N-linked glycans at Asn positions 280, 302, 399 and 406 [48]. None of
N‐linkage was initially obtained from the X‐ray crystal structure of the protein. The structure revealed these Asn that the protein had four N‐linked glycans at Asn positions 280, 302, 399 and 406 [48]. None of these were located in an Asn-X-(Thr/Ser) sequon sequence commonly recognized by ER located
Asn were located in an Asn‐X‐(Thr/Ser) sequon sequence commonly recognized ER located glycosyltransferases
[50–52].
This
finding also
explained
why
prior attempts
toby remove
Vp54 glycans
glycosyltransferases [50‐52]. This finding also explained why prior attempts to remove Vp54 glycans with enzymes that cleave traditional N-linked glycans were unsuccessful [53] Nandhagopal et al. [48]
with enzymes that cleave traditional N‐linked glycans were unsuccessful [53] Nandhagopal et al. [48] also reported
Vp54
contained
two O-linked
glycans.
However,
the
X-ray crystal
also that
reported that Vp54 contained two O‐linked glycans. However, re-examination
re‐examination of of
the X‐ray crystal data (Figure 3) indicates that no O‐linked glycans are present in the protein [49], which agrees data (Figure
3) indicates that no O-linked glycans are present in the protein [49], which agrees with
with our unsuccessful attempts to detect them by chemical procedures. our unsuccessful
attempts to detect them by chemical procedures.
Figure 3. Figure 3. Structure of the revised PBCV‐1 Vp54 monomer. The two jelly‐roll domains are colored in green Structure of the revised PBCV-1 Vp54 monomer. The two jelly-roll domains are colored
and red, respectively. The glycans located on the surface are shown as a space‐filling representation of their in green and
red, respectively. The glycans located on the surface are shown as a space-filling
atoms and are colored according to the residue they are attached to (Asn‐280: green, Asn‐302: black, representation
of their atoms and are colored according to the residue they are attached to (Asn-280:
Asn‐399: red, Asn‐406: blue). Taken from De Castro et al. [49] with permission. green, Asn-302: black, Asn-399: red, Asn-406: blue). Taken from De Castro et al. [49] with permission.
5. Glycan Structures Attached to Chlorovirus Major Capsid Proteins The structures of the PBCV‐1 Vp54 N‐linked glycans were reported recently, and they consist of 8–10 neutral monosaccharide residues, producing a total of four glycoforms (Figure 4) [54]. These structures do not resemble any structure previously reported in the three Domains of Life. Among Viruses 2017, 9, 88
11 of 23
5. Glycan Structures Attached to Chlorovirus Major Capsid Proteins
The structures of the PBCV-1 Vp54 N-linked glycans were reported recently, and they consist of
8–10
neutral monosaccharide
residues, producing a total of four glycoforms (Figure 4) [54]. 11 of 23 These
Viruses 2017, 9, 88 structures do not resemble any structure previously reported in the three Domains of Life. Among
their
most distinctive features are: (1) the four glycoforms share a common core structure, and the four
their most distinctive features are: (1) the four glycoforms share a common core structure, and the glycoforms
are related to the non-stoichiometric presence of two monosaccharides, L-arabinose
and
four glycoforms are related to the non‐stoichiometric presence of two monosaccharides, L‐arabinose D-mannose;
the most
glycoform
consists consists of nine neutral
monosaccharide
residues organized
and D‐mannose; the abundant
most abundant glycoform of nine neutral monosaccharide residues in
a highly-branched fashion; (2) the glycans are attached to the protein by a β-glucose linkage,
organized in a highly‐branched fashion; (2) the glycans are attached to the protein by a β‐glucose linkage, which
is rare in nature and has only been reported in glycoproteins from a few organisms [55–58];
which is rare in nature and has only been reported in glycoproteins from a few organisms [55–58]; and
the glycoform glycoform contains containsa adimethylated dimethylatedrhamnose rhamnoseas asthe thecapping cappingresidue residue
themain main
chain,
and (3)
(3) the of ofthe chain, a ahyper‐branched fucose residue and two rhamnose residues with opposite absolute configurations. hyper-branched fucose residue and two rhamnose residues with opposite absolute configurations.
Figure 4.
4. Structures of PBCV-1
PBCV‐1 Vp54
Vp54 N-glycans.
N‐glycans. Arabinose mannose are
are not
not stoichiometric
stoichiometric Figure
Structures of
Arabinose and and mannose
substituents and create four different glycoforms. The two on the left are the most abundant, and both substituents
and create four different glycoforms. The two on the left are the most abundant, and
have have
mannose. The The
structure at the bottom represents the is both
mannose.
structure
at the
bottom
represents
theconserved conservedcore coreoligosaccharide oligosaccharide that that is
present in all of the chloroviruses studied to date. Residues within the box are those strictly conserved, present
in all of the chloroviruses studied to date. Residues within the box are those strictly conserved,
while rhamnose (outside the box) is a semi‐conserved element because its absolute configuration is while
rhamnose (outside the box) is a semi-conserved element because its absolute configuration is
virus dependent. The figure was modified from De Castro et al. [34,54] with permission. virus
dependent. The figure was modified from De Castro et al. [34,54] with permission.
Attempts to fit the Vp54 glycan structures into the original Vp54 X‐ray crystal structure [48] were Attempts
to fit the Vp54 glycan structures into the original Vp54 X-ray crystal structure [48] were
unsuccessful and led to a re‐examination of the original structure. This re‐examination produced a unsuccessful and led to a re-examination of the original structure. This re-examination produced a
structure that was compatible with the four N‐linked glycan structures (Figure 3) [49]. As mentioned structure
that was compatible with the four N-linked glycan structures (Figure 3) [49]. As mentioned
above, the revised structure lacks the two O‐linked glycans reported originally. above, the revised structure lacks the two O-linked glycans reported originally.
The PBCV‐1 Vp54 has a molecular weight of 53,790 Da. The a430l gene encodes Vp54 with a The
PBCV-1 Vp54 has a molecular weight of 53,790 Da. The a430l gene encodes Vp54 with a
predicted molecular weight of 48,165 Da so the combined sugars have a molecular weight of 5625 Da, predicted molecular weight of 48,165 Da so the combined sugars have a molecular weight of 5625 Da,
which is about the weight of the four glycans. Vp54 was also reported to have a myristic acid attached which
is about the weight of the four glycans. Vp54 was also reported to have a myristic acid attached
to the carboxyl portion of the protein [59]. However, myristic acid has not been observed in any of to the carboxyl portion of the protein [59]. However, myristic acid has not been observed in any of
the recent Vp54 structural experiments, and so, its status is currently unknown. The structures of the the
recent Vp54 structural experiments, and so, its status is currently unknown. The structures of the
Vp54 glycans from the PBCV‐1 antigenic variants, referred to above, are currently being determined, Vp54 glycans from the PBCV-1 antigenic variants, referred to above, are currently being determined,
and as expected, the structures are truncated forms of the wild‐type PBCV‐1 glycans [44]. PBCV‐1 and
as expected, the structures are truncated forms of the wild-type PBCV-1 glycans [44]. PBCV-1
particles were reported to have two additional glycoproteins in addition to Vp54 [59]. Both of these particles
were reported to have two additional glycoproteins in addition to Vp54 [59]. Both of these
glycoproteins react
react with
with the PBCV‐1 antibody, so, glycan
the glycan structures are predicted to be glycoproteins
the
PBCV-1
antibody,
andand so, the
structures
are predicted
to be similar
similar or identical to the glycans associated with Vp54. gene encoding one of these proteins or
identical
to the glycans
associated
with Vp54.
The
geneThe encoding
one of these
proteins
(Vp260)
(Vp260) was identified (gene a122r). Gene a122r homologs are common in the chloroviruses, and some of the viruses have as many as five copies of the gene [60]. The role that Vp260 plays in the PBCV‐1 virion is unknown. The glycan structures of the major capsid proteins from seven more chloroviruses, which represent all four chlorovirus types, were recently reported (Figure 5) [6,34,35]; collectively, all of the Viruses 2017, 9, 88
12 of 23
was identified (gene a122r). Gene a122r homologs are common in the chloroviruses, and some of the
viruses have as many as five copies of the gene [60]. The role that Vp260 plays in the PBCV-1 virion
is unknown.
The glycan structures of the major capsid proteins from seven more chloroviruses, which represent
all four chlorovirus types, were recently reported (Figure 5) [6,34,35]; collectively, all of the glycans have
a common
core region
(outlined in Figure 4). The common core region consists of a pentasaccharide
Viruses 2017, 9, 88 12 of 23 with a β-glucose linked to an Asn residue, which is not located in the typical sequon Asn-X-(Thr/Ser).
The
glucose has a terminal xylose unit and a hyperbranched fucose, which is in turn substituted
Asn‐X‐(Thr/Ser). The glucose has a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose with
a terminal galactose and a second xylose residue. The third position of the fucose unit is
unit is always linked to a rhamnose, which is a semi‐conserved element because its configuration is always
linked to a rhamnose, which is a semi-conserved element because its configuration is virus
virus dependent. Additional decorations occur this core N‐glycan and represent a molecular dependent.
Additional
decorations
occur on
this on core
N-glycan
and represent
a molecular
signature
signature for each chlorovirus. for each chlorovirus.
Figure Structures ofof N-glycans
N‐glycans from
from seven
seven chloroviruses
chloroviruses representing all allfour chlorovirus types. Figure
5. 5. Structures
representing
four
chlorovirus
types.
Substituents in brackets are not stoichiometric. All sugars are in the pyranose form, except where Substituents in brackets are not stoichiometric. All sugars are in the pyranose form, except where
specified. Virus NY‐2A is an NC64A virus, virus Osy‐NE5 an Osy virus, viruses ATCV‐1 and TN603 SAG specified.
Virus NY-2A is an NC64A virus, virus Osy-NE5 an Osy virus, viruses ATCV-1 and TN603
MT325, CVM‐1 and NE‐JV‐1 Pbi viruses. This figure modified from [6,34,35] with viruses and and
SAG
viruses
MT325,
CVM-1
and NE-JV-1
Pbi viruses.
This was figure
was modified
from [6,34,35]
permission. with
permission.
6. Chlorovirus PBCV‐1 Encoded Glycosyltransferases In addition to the two glycosyltransferases, hyaluronan synthase and chitin synthase previously described, the 43 chloroviruses collectively encode eight putative glycosyltransferases (Table 4). Six of these eight glycosyltransferase‐encoding genes are in PBCV‐1; they are scattered throughout the PBCV‐1 genome. None of these six PBCV‐1 encoded glycosyltransferases have an identifiable signal Viruses 2017, 9, 88
13 of 23
6. Chlorovirus PBCV-1 Encoded Glycosyltransferases
In addition to the two glycosyltransferases, hyaluronan synthase and chitin synthase previously
described, the 43 chloroviruses collectively encode eight putative glycosyltransferases (Table 4). Six of
these eight glycosyltransferase-encoding genes are in PBCV-1; they are scattered throughout the PBCV-1
genome. None of these six PBCV-1 encoded glycosyltransferases have an identifiable signal peptide
that would target them to the ER. Furthermore, with the exception of PBCV-1 glycosyltransferases
A473L (six transmembrane domains; CESA CelA-like) and A219/222/226R (nine transmembrane
domains; CXCX-2), none of the four remaining PBCV-1 encoded glycosyltransferases are predicted to
have transmembrane domains. Therefore, these enzymes are expected to be soluble proteins. The genes
for the six PBCV-1 encoded glycosyltransferases are expressed early during PBCV-1 infection [61].
Thus, assuming the enzymes are stable, they would be available for adding sugars to the Vp54 glycans
during virus replication.
The PBCV-1 a064r gene encodes a 638-amino acid protein with three predicted domains.
The N-terminal 211 amino acid domain resembles a “fringe-class” of glycosyltransferases (GT-GTA) and
contains the last four of the five conserved motifs characteristic of this group of glycosyltransferases [62,63],
including the proposed catalytic amino acids, the Asp-X-Asp sequence in motif 3 and the first Asp
residue in motif 5. However, spacing between some of the four motifs differs from that of the
fringe-glycosyltransferases. As mentioned above, the A064R protein, which is only present in five
NC64A viruses, lacks both an identifiable signal peptide that would target the protein to the ER and a
membrane-spanning motif, in contrast to “fringe” glycosyltransferases.
The 211-amino acid A064R glycosyltransferase domain was cloned, and the recombinant protein
was crystallized [64]. The 1.6 Å crystal structure of the peptide has a mixed α/β fold containing a
central, six-stranded β sheet flanked by α helices. The overall fold is similar to the catalytic domains
in retaining glycosyltransferases in the GT-A group, family 34, although the amino acid similarity
between them is low. Zhang et al. [64] suggested that the A064R glycosyltransferase bound to
UDP-glucose better than to UDP-galactose or UDP-N-acetyl glucosamine. However, these binding
experiments were conducted prior to knowing the Vp54 glycan structures. Now, there is evidence that
the glycosyltransferase domain adds L-rhamnose to the distal xylose residue in the core structure [45].
Analysis of the six PBCV-1 antigenic variants revealed mutations in a064r that correlated with
a specific antigenic class, B (EPA-1) (Table 3). The a064r gene in all six of these antigenic variants
was sequenced to determine if mutations in a064r correlated with the EPA-1 antigenic variation [46].
The a064r sequences from three of the mutants had single nucleotide substitutions, which produced
a single amino acid substitution in the glycosyltransferase portion of the A064R protein. Two of
the amino acid substitutions occurred in the Asp-X-Asp motif (domain 3), and the other one was in
domain 4. A fourth variant had an extra base in the coding sequence, which created a frame shift
mutation in the gene. Finally, the entire gene was deleted in the other two antigenic variants.
Dual infection experiments with some of the different antigenic variants established that viruses
containing wild-type a064r complemented and recombined with viruses that contained variant a064r
to form wild-type virus. Therefore, it was concluded that a064r encodes a glycosyltransferase involved
in the synthesis of the Vp54 glycan [46].
As noted above, the protein product of the a064r gene contains three domains with domain 1 being
the glycosyltransferase. Domain 2 does not match anything in GenBank, but the C-terminal domain 3
is predicted to be a methyltransferase. We suspect that this C-terminal domain of approximately
200 amino acids is involved in methylating the terminal L-rhamnose in the Vp54 glycan [45].
A homolog of PBCV-1 glycosyltransferase, A546L (GT-GT4), has also been produced and
crystallized [65]. The a546l gene homolog was from another NC64A chlorovirus NY-2A (gene b736l),
and the 396-amino acid protein resembles members in the GT4 family of glycosyltransferases in the
CAZy classification [66,67]. However, its biochemical function remains to be elucidated.
Viruses 2017, 9, 88
14 of 23
Table 4. Chlorovirus encoded enzymes involved in synthesizing glycans attached to virus major capsid proteins.
Host
Viruses
EXT 1
GT-A 2
GT-GT4 3
GT-GTA 4
CESA CelA-Like 5
CSCS-2 6
078L
C093L
080L
039L
071L
175R
024L
531R
533R
085L
116L
107L
098L
A075L
123R
C150R
117R
077R
120R
109L
060R
4549L
128R
124R
180R
B159R
162R
A111/114R
559R
C661L
594L
611L
606L
773L
528L
753L
702L
631L
754L
B736L
760L
A546L
065R
104R
060R
518L
535L
099R
NC64A
AN69C
AR158
CviK1
CvsA1
IL-3A
IL-5-2s1
KS-1B
MA-1D
MA-1E
NE-JV-4
NY-2B
NY2A
NYs-1
PBCV-1
009R
046L
074R
626L
108L
160R
255R
C265R
242R
247R
240R
313R
170R
194R
281R, 210R
250R
323R
SYN
OSY-NE-5
025L
044R
283L
Pbi
AP110A
CVA-1
CVB-1
CVG-1
CVM-1
CVR-1
CZ-2
Can18-4
FR483
FR5L
MT325
NE-JV-1
NW665
OR0704.2.2
013L
016L
025L
019L
022L
020L
012L
020L
N012L
046L
M009L
079L
015L
017L
548R
532R
538R
520R
550R
545R
532R
557R
N472R
537R
M467R
464L
532R
549R
A064R
A473L
649R
637R
633R
B618R
641R
187R
A219/222/226R
380L
229R
N191R
M186R
811L
815L
822L
862L
N715L
819L
M721L
801R
849L
823L
GT17 8
C559R
097L
226R
220R
232R
217R
237R
225R
382L
GT 7
970R
971R
918R
920R
953R
977R
932R
971R
N805R
926R
M813R
930R
955R
923R
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Table 4. Cont.
1
Host
Viruses
EXT 1
GT-A 2
GT-GT4 3
Z830R
959R
1007R
874R
977R
1009R
932R
1020R
970R
1044R
1006R
991R
951R
Z120R
137R
140R
141R
141R
165R
138R
150R
145R
156R
146R
141R
141R
Z667L
SAG
ATCV-1
Br0604L
Can0610SP
Canal-1
GM0701
MN0810
MO0605SPH
NE-JV-2
NE-JV-3
NTS-1
OR0704.3
TN603
WI0606
789L
164L
752L
164L
804L
778L
944L
GT-GTA 4
CESA CelA-Like 5
CSCS-2 6
GT 7
Z178L, Z823R, Z417L
225L, 952R, 483L
210L, 1002R, 978L, 487L
871R
228L, 975R, 486L
244L
230L, 926R, 479L
1015R, 992L, 516L
215L, 210L, 964R, 484L
1016L, 516L
1001R, 972L, 485L, 219L
226L, 986R, 475L
233L, 945R, 506L
Z425R
489R
495R
447R
493R
Z347R
399R
407R
380R
405R
424R
412R
438R
407R
441R
404R
400R
430R
488R
523R
493R
493R
483R
514R
GT17 8
Exotosin glycosyltransferase; 2 glycosyltransferase family A; 3 glycosyltransferase GT4-type super family; 4 glycosyltransferase GTA-type super family; 5 CESA CelA-like cellulose
synthase catalytic subunit (UDP-glucose as substrate); 6 cellulose synthase catalytic subunit; 7 glycosyltransferase; 8 glycosyltransferase family 17.
Viruses 2017, 9, 88
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Of the eight glycosyltransferases encoded by the 43 chloroviruses, only two of them, homologs
of PBCV-1 A111/114R and A075L, are present in all of the viruses, and so, they are predicted to be
involved in the synthesis of the core glycan structure. A111/114R is especially interesting because it is
predicted to have at least two glycosyltransferase catalytic domains.
Now that structures of the glycans from the chlorovirus major capsid proteins are becoming
available, one can begin to characterize the viral encoded glycosyltransferases biochemically.
One question that needs to be addressed is: Are the sugars added sequentially to the Vp54 protein
backbone or are the glycans initially synthesized independently of Vp54, possibly on a lipid carrier
and then attached to the protein in a single step? A slight variation of these two possibilities is that
the core glycan is synthesized independently of the protein and then attached to Vp54. Additional
sugars could then be added sequentially to these core glycans [68]. We suspect that this viral encoded
glycosylation pathway represents a previously undescribed pathway, possible even a pathway that
existed in eukaryotes prior to the ER and Golgi glycosylation pathway [18].
7. Additional Chlorovirus Encoded Sugar Metabolism Enzymes
Besides the chlorovirus-encoded enzymes described above, the viruses have four additional
genes predicted to encode enzymes involved in sugar metabolism (Table 2). Recombinant proteins
have not been produced from any of these genes, and so, it is unknown if they encode functional
enzymes. These putative enzymes include an acetyltransferase (AT) encoded by all 43 chloroviruses,
a D-lactate dehydrogenase (D-LD) encoded by 32 chloroviruses, fumarate reductase (FRD) encoded by
five chloroviruses and ADP-ribosyl glycohydrolase (ADP-RGH) encoded by nine chloroviruses, all but
two of which are Pbi viruses. The roles these putative enzymes play in the viral life cycles are unknown.
8. Chlorovirus-Encoded Polysaccharide Degrading Enzymes
In addition to the polysaccharide synthesizing enzymes described above, the chloroviruses also
encode polysaccharide-degrading enzymes (Table 5). The chloroviruses are unique among viruses
infecting eukaryotic organisms in that they, like bacteriophages, need to penetrate a rigid algal cell
wall to initiate infection. The icosahedral shaped chlorovirus PBCV-1 has a spike-like structure at
one vertex [13], which appears to make the initial contact with the cell wall of its host, C. variabilis
NC64A [69]. Attachment is immediately followed by host cell wall degradation at the point of contact
by a virus-packaged enzyme(s) [70]. After wall degradation, the viral internal membrane fuses with
the host membrane to produce a narrow (~5 nm in inner diameter), membrane-lined tunnel, which
allows entry of the viral DNA and some viral proteins [71]. This membrane fusion results in immediate
host membrane depolarization [72] and potassium ion efflux [73]. This process results in an empty
capsid remaining on the host cell surface.
Table 5. Chlorovirus encoded enzymes involved in degrading polysaccharides.
Host
Viruses
CHI 1
CHIS 2
GUN 3
BCHIL 4
Lysin 5
AN69C
AR158
CviK1
297R
331L
C342L
309L
102L
C126L
99L
204R
C220R
194R
540R
C681L
610L
318L
058L
200R
627L
323L
415L
257L
097L
140R
048R
196R
262R
132R
624L
796L
546L
CvsA1
NC64A
IL-3A
IL-5-2s1
KS-1B
285R
373R
211R
241R,
242R
MA-1D
MA-1E
NE-JV-4
NY-2B
NY2A
NYs-1
PBCV-1
SYN
OSY-NE-5
298R
367R
a
360R
A260R
119R
GH 6
CD 7
ALGL 8
387L
C415L
362L
371L,
373L
378L
495L
250L
C263L
237L
243L
235L
310L
166L
283L
491R
147R
777L
359L
191L
352L
337L
406L
B393L
403L
a
A292L
114R
110R
158L
B137L
134L
a
A094L
194R, 229R
205R
270R
B239R
251R
a
A181/182R
717L
648L
777L
B756L
399L
277L
246L
319L
B288L
138L
037L
117L
a
A561L
290L
476L
B469L
487L
a
A215L
099R
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Table 5. Cont.
Host
Viruses
CHI 1
CHIS 2
GUN 3
BCHIL 4
Lysin 5
106R
102R
129R
105R
122R
109R
070R
113R
122R
114R
143R
113R
133R
121R
079R
121R
N087R
106R
M091R
218L
088R
082R
174L
167L
942R
942R
890R
896R
927R
948R
902R
944R
N779R
897R
M791R
088R
921R
895R
306R
297R
310R
303R
330R
Pbi
AP110A
CVA-1
CVB-1
CVG-1
CVM-1
CVR-1
CZ-2
Can18-4
FR483
FR5L
MT325
NE-JV-1
NW665
OR0704.2.2
263R
316R
N262R
300R
M258R
592L
281R
257R
Z819L
Z814L
Z511L
950L
902R, 942L
518L
ATCV-1
098R
M085R
328L
074R
Z780L
Br0604L
Can0610SP
SAG
Canal-1
GM0701
MN0810
MO0605SPH
Z204R
253R,
254R
244R
815L
871L
242R
251R
271R
257R
NE-JV-2
950L
258R
NE-JV-3
NTS-1
907L
973L
244R
276R
OR0704.3
251R
TN603
WI0606
253R
262R
889L
142L
184L
174L
114L
160L
N119L
M124L
275L
128L
113L
985R,
1001L
866L
972L
082L
920L
1000R,
1013L
957L
1024R
979R,
999L
983L
938L
936R, 996R
613L
138L, 855L
917R, 963L
162L, 1003L
911L
546L
526L
659L
588L
GH 6
CD 7
ALGL 8
158L
156L
183L
338R
327R
342R
329R
360R
335R
293R
349R
N293R
336R
M289R
472L
315R
292R
171L
163L
157L
050L
269L
Z771L
832L
895L
743L
846L
896L
806L
910L
959L
862L
919L
1008R
634L
932L
948L
1039R
603L
556L
897L
961L
918R, 994R
604L
939R, 977L
929L
520L
612L
886L
910L
867L
931L
878L
1
Chitinase; 2 chitosanase; 3 1-3-beta glucanase; 4 bifunctional chitinase/lysozyme; 5 lysin homologs from encoded
by PBCV-1 CDS A561L; 6 glycosyl hydrolase; 7 chitin deacetylase; 8 alanine alginate lyase; a the recombinant proteins
have the predicted activity.
In addition to virus entry into the host cells, nascent infectious PBCV-1 viruses exit the cells at
6–8 h PI by lysis of the plasma membrane and the cell wall. Therefore, it is not surprising that the
chloroviruses encode polysaccharide-degrading enzymes in order to enter and exit the host cell. In fact,
PBCV-1 encodes five such enzymes (Table 5), including two chitinases [74,75], a chitosanase [74,76],
a β-1,3 glucanase [77] and an alkaline alginate lyase [78] or a polysaccharide lyase, cleaving chains
of β- or α-1,4-linked glucuronic acids [79,80]. Recombinant proteins have been produced from each
of these genes and shown to have the expected activity. Interestingly, the β-1,3 glucanase gene is
expressed very early and disappears by 60 min PI. The protein is also made very early and disappears
by 90 min PI [77]. Therefore, this enzyme is unlikely to be involved in either viral entry or viral exit
from the cell. One possible function for the enzyme is to degrade host β-1,3 glycans, which might
serve as host storage polysaccharides. Gene transcripts from the other four polysaccharide-degrading
enzymes are present throughout the viral life cycles [74,81].
Experiments conducted about 30 years ago established that a crude enzyme preparation made
from PBCV-1 lysates, named lysin, had good wall degrading activity and could be used to produce
C. variabilis NC64A protoplasts [82,83]. Therefore, it was assumed that one or more of the five PBCV-1
encoded enzymes would be packaged in the PBCV-1 virion and be responsible for degrading the host
cell wall at the point of infection. In fact, Yamada et al. [76] reported that a chitosanase activity was
packaged in a closely-related chlorovirus, CVK2. However, an ensuing report [75] indicated that the
CVK2 chitosanase activity was due to incomplete purification of the virion. Subsequently, a PBCV-1
proteome study identified 148 virus-encoded proteins and one host-encoded protein in highly purified
virions [10]. Surprisingly, none of the five polysaccharide-degrading enzymes were packaged in the
PBCV-1 virions.
Consequently, the 148 virus-encoded proteins packaged in the PBCV-1 particles were re-examined
for possible polysaccharide or cell wall degrading activity. This effort revealed that one of the
PBCV-1-encoded proteins packaged in the virion, A561L, has a putative glycosyl hydrolase domain.
Viruses 2017, 9, 88
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A recombinant protein produced from this domain has cell wall degrading activity, and the protein is
under active investigation [84]. Homologs of the A561L domain (named A561L lysin) are present in
most of the chloroviruses (Table 5), but not all. For example, viruses NYs-1 and CVR-1 appear to lack
an a561l gene homolog encoding this domain, and the similarity between the predicted A561L homolog
from viruses NY-2B and WI0606 is not very high. The apparent absence of the protein from these
viruses deserves to be investigated further because one would expect the enzyme(s) that degrades the
host cell wall during virus infection would be highly conserved.
Twenty-four of the 43 chloroviruses encode a protein that has a polysaccharide deacetylase
domain (Table 5). Viruses in three of the four types (Osy being the exception) have the gene, but
it is also missing in some viruses in each of the three types, so the gene is clearly not required for
the success of the viruses. Its role might be to remove the acetyl group from chitin during host cell
wall degradation.
In addition to these glycolytic enzymes, 42 of the 43 chloroviruses, encode a functional glycosylase
protein that initiates pyrimidine photodimer excision [85,86]. The enzyme is part of a DNA repair pathway.
9. Conservation of the Chlorovirus Encoded Sugar Enzymes
Only three of the 21 chlorovirus encoded proteins listed in Tables 1, 2 and 4 are present in all
43 chloroviruses, and these would be considered to be core proteins. The three are an acetyltransferase
(AT), an exostosin glycosyltransferase (EXT) and a family A glycosyltransferase (GT-A). As noted
above, we predict that the two glycosyltransferases are involved in the synthesis of the glycan core
attached to the major capsid protein. The predicted function of the acetyltransferase is unknown.
Two of the seven viral encoded proteins involved in polysaccharide degrading activity are
conserved in all of the viruses, the chitosanase (CHIS) and a putative bifunctional chitinase/lysozyme
(BCHIL) (Table 4). Presumably these two enzymes play a role in the release of the nascent viruses from
the cell. As indicated above, they are not packaged in the PBCV-1 virion, and so, they are not involved
in the immediate early virus infection process.
The presence or absence of some of the chlorovirus sugar encoding enzymes displays some
interesting patterns. For example, the GMD and GMER encoding genes are present in all of the NC64A,
SAG and Osy viruses and absent in all of the Pbi viruses. This observation would suggest that the
three virus types that have these genes would be more closely related to each other than to the Pbi
viruses. However, a phylogenetic tree that shows the evolutionary relationship between the 43 viruses
based on 29 concatenated core proteins [6] indicates that SAG and Pbi viruses are in the same branch
and that the NC64A and Osy viruses are in a separate branch. Therefore, these results would imply
that the SAG and NC64A/Osy viruses either acquired the genes separately after the four virus types
had separated from a common ancestor or that the chlorovirus ancestor had both genes and for some
reason, they were lost in the Pbi lineage.
Most of the other protein patterns are even more difficult to explain. For example, the GFAT
encoding gene is present in all of the Pbi viruses and present in 11 of the 14 NC64A viruses and one
SAG virus. Several of the other genes have similar complicated patterns and await explanations.
10. Sugar Enzymes Coded by Other Large DNA Viruses
This review has focused on carbohydrate enzymes encoded by the chloroviruses, primarily
because these enzymes have been the most intensively studied. However, as new giant viruses are
being discovered and their genomes sequenced, it is clear that some of them encode putative enzymes
involved in carbohydrate manipulations. The most extensively studied of these other large DNA
viruses is Acanthamoeba polyphaga mimivirus, which has genes encoding both glycosyltransferases
and nucleotide sugars (see the recent review by Piacente et al. [87]). Other large DNA viruses
encoding putative sugar manipulating enzymes include prasinoviruses (family Phycodnaviridae like the
chloroviruses) that infect small marine green algae, including Ostreococcus, Bathycoccus and Micromonas
species; these viruses have clusters of putative genes for enzymes involved in nucleotide-sugar
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metabolism and glycosyltransferases [88]. Similar genes are present in other members of the
Mimiviridae family, including Phaeocystis globosa virus [89] and Cafeteria roenbergensis virus [90]. Putative
glycosyltransferase-encoding genes have also been reported in the genomes of pandoraviruses [91],
Pithovirus sibericus [92] and Mollivirus sibericus [93].
In conclusion, it is becoming clear that virus-encoded sugar-manipulating enzymes and
glycosylation systems can no longer be considered a hallmark solely of cellular organisms, but that
some viruses also encode unique and complex glycan systems, which are still largely unknown.
One encourages young glycobiologists to consider working on some of these systems.
Acknowledgments: The authors thank the many undergraduate students, graduate students, postdocs and
collaborators who have worked on various aspects of the chlorovirus encoded sugar enzymes over the past
25 years. This research has been supported by grants from NIH, NSF, DOE and the Stanley Medical Research
Institute over the years.
Conflicts of Interest: The authors declare no conflict of interest.
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